1. Field of the Invention
The present invention concerns the construction of a protein having a reduced or eliminated ability to bind human IgA, but that retains the immunological properties useful for formulating a conjugate vaccine against Group B streptococci.
2. Related Art
Streptococci are a large and varied set of gram-positive bacteria which have been ordered into several groups based on the antigenicity and structure of their cell wall polysaccharide (Lancefield, R. C., J. Exp. Med. 57:571-595 (1933); Lancefield, R. C., Proc. Soc. Exp. Biol. and Med. 38:473-478 (1938)). Two of these groups have been associated with serious human infections. Those that have been classified into Group A streptococci are the bacteria that people are most familiar and are the organisms which cause "strep throat." Organisms of Group A streptococci also are associated with the more serious infections of rheumatic fever, streptococcal impetigo, and sepsis.
Group B streptococci were not known as a human pathogen in standard medical textbooks until the early 1970's. Since that time, studies have shown that Group B streptococci are an important perinatal pathogen in both the United States as well as the developing countries (Smith, A. L. and J. Haas, Infections of the Central Nervous System, Raven Press, Ltd., New York. (1991) p. 313-333). Systemic Group B streptococcal infections during the first two months of life affect approximately three out of every 1000 births (Dillon, H. C., Jr., et al., J Pediat. 110:31-36 (1987)), resulting in 11,000 cases annually in the United States. These infections cause symptoms of congenital pneumonia, sepsis, and meningitis. A substantial number of these infants die or have permanent neurological sequelae. Furthermore, these Group B streptococcal infections may be implicated in the high pregnancy-related morbidity which occurs in nearly 50,000 women annually. Others who are at risk from Group B streptococcal infections include those who either congenitally, chemotherapeutically, or by other means, have an altered immune response.
Group B streptococci can be further classified into several different types based on the bacteria's capsular polysaccharide. The most pathogenically important of these different types are streptococci having types Ia, Ib, II, or III capsular polysaccharides. Group B streptococci of these four types represent over 90% of all reported cases. The structure of each of these various polysaccharide types has been elucidated and characterized (Jennings, H. J., et al., Biochemistry 22:1258-1263 (1983); Jennings, H. J., et al., Can. J. Biochem. 58:112-120(1980); Jennings, H. J., et al., Proc. Nat. Acad. Sci. USA. 77:2931-2935 (1980); Jennings, H. J., et al., J. Biol. Chem. 258:1793-1798 (1983); Wessels, M. R., et al., J. Biol. Chem. 262:8262-8267 (1987)). As is found with many other human bacterial pathogens, it has been ascertained that the capsular polysaccharides of Group B streptococci, when used as vaccines, provide very effective, efficacious protection against infections with these bacteria. This was first noted by Lancefield (Lancefield, R. C., et al., J. Exp. Med. 142:165-179 (1975)) and more recently in the numerous studies of Kasper and coworkers (Baker, C. J., et al., N. Engl. J. Med. 319:1180-1185 (1988); Baltimore, R. S., et al., J. Infect. Dis. 140:81-86 (1979); Kasper, D. L., et al., J. Exp. Med. 149:327-339 (1979); Madoff, L. C., et al., J. Clin. Invest. 94:286-292 (1994); Marques, M. B., et al., Infect. Immun. 62:1593-1599 (1994); Wessels, M. R., et al., J. Clin. Invest. 86:1428-1433 (1990); Wessels, M. R., et al., Infect. Immun. 61:4760-4766 (1993); Wyle, S. A., et al., J. Infect. Dis. 126:514-522 (1972)). However, much like many other capsular polysaccharide vaccines (Anderson, P., et al., J. Clin. Invest. 51:39-44 (1972); Gold, R., et al., J. Clin. Invest. 56:1536-1547 (1975); Gold, R., et al., J. Infect. Dis. 136S:S31-S35 (1977); Gold, R. M., et al., J. Infect. Dis. 138:731-735 (1978); Makela, P. R. H., et al., J. Infect. Dis. 136:S43-50 (1977); Peltola, A., et al., Pediatrics 60:730-737 (1977); Peltola, H., et al. N. Engl. J. Med. 297:686-691 (1977)), vaccines formulated from pure type Ia, Ib, II, and III capsular carbohydrates are relatively poor immunogens and have very little efficacy in children under the age of 18 months (Baker, C. J. and D. L. Kasper. Rev. Inf. Dis. 7:458-467 (1985); Baker, C. J., et al., N. Engl. J. Med. 319:1180-1185 (1988); Baker, C. J., et al., New Engl. J. Med. 322:1857-1860 (1990)). These pure polysaccharides are classified as T cell independent antigens because they induce a similar immunological response in animals devoid of T lymphocytes (Howard, J. G., et al., Cell. Immunol. 2:614-626 (1971)). It is thought that these polysaccharides do not evoke a secondary booster response because they do not interact with T cells, and therefore fail to provoke a subsequent "helper response" via the secretion of various cytokines. For this reason, each consecutive administration of the polysaccharide as a vaccine results in the release of a constant amount of antibodies, while a T cell dependent antigen would elicit an ever increasing concentration of antibodies each time it was administered.
Goebel and Avery found in 1931 that by covalently linking a pure polysaccharide to a protein that they could evoke an immune response to the polysaccharide which could not be accomplished using the polysaccharide alone (Avery, O. T. and W. F. Goebel, J. Exp. Med. 54:437-447 (1931); Goebel, W. F. and O. T. Avery, J. Exp. Med. 54:431-436 (1931)). These observations initiated and formed the basis of the current conjugate vaccine technology. Numerous studies have followed and show that when polysaccharides are coupled to proteins prior to their administration as vaccines, the immune response to the polysaccharides changes from a T independent response to a T dependent response (see Dick, W. E., Jr. and M. Beurret, Glycoconjugates of bacterial carbohydrate antigens In: Contributions to Microbiology and Immunology. Cruse et al., eds., (1989) p. 48-114; Jennings, H. J. and R. K. Sood, Neoglycoconjugates: Preparation and Applications. Y. C. Lee and R. T. Lee, eds., Academic Press, New York. (1994) p. 325-371; Robbins, J. B. and R. Schneerson, J. Infect. Dis. 161:821-832 (1990)for reviews). Currently, most of these polysaccharide-protein conjugate vaccines are formulated with well known proteins such as tetanus toxoid and diphtheria toxoid or mutants thereof. These proteins were originally used because they were already licensed for human use and were well characterized. However, as more and more polysaccharides were coupled to these proteins and used as vaccines, interference between the various vaccines which used the same protein became apparent. For example, if several different polysaccharides were linked to tetanus toxoid and given sequentially, the immune response to the first administered polysaccharide conjugate would be much larger than the last. If, however, each of the polysaccharides were coupled to a different protein and administered sequentially, the immune response to each of the polysaccharides would be the same. Carrier suppression is the term used to describe this observed phenomenon. One approach to overcome this problem is to match the protein and polysaccharide so that they are derived from the same organism.
Among the various antigens used to classify and subgroup Group B streptococci, one was a protein known as the Ibc antigen. This protein antigen was first described by Wilkinson and Eagon in 1971 (Wilkinson, H. W. and R. G. Eagon, Infect. Immun. 4:596-604 (1971)) and was known to be made up of two distinct proteins designated as alpha and beta. Later, the Ibc antigen was shown to be effective when used as a vaccine antigen in a mouse model of infection by Lancefield and co-workers (Lancefield, R. C., et al., J. Exp. Med. 142:165-179 (1975)). The isolation, purification and functional characterization of the beta antigen (C.beta.) protein of Group B streptococci was accomplished by Russell-Jones, et al. (Russell-Jones, G. J. and E. C. Gotschlich, J. Exp. Med. 160:1476-1484 (1984); Russell-Jones, G. J., et al., J. Exp. Med. 160:1467-1475 (1984))(see U.S. Pat. No. 4,757,134)). They could demonstrate that one of the properties of the C.beta. protein was to bind specifically to human IgA immunoglobulin. The binding site on the IgA molecule was localized to the Fc portion of the heavy chain of this immunoglobulin. They further showed that the C.beta. protein consisted of a single polypeptide having an estimated molecular weight of 130,000 daltons. The gene responsible for the expression of the C.beta. protein was cloned (Cleat, P. H. and K. N. Timmis, Infect. Immun. 55:1151-1155 (1987)) and sequenced (Jerlstrom, P. G., et al., Molec. Microbiol. 5:843-849 (1991)) by a group led by Timmis. His later study demonstrated that the IgA binding activity could be assigned to a 746 bp DNA fragment of the gene defined by a leading BglII restriction endonuclease cleavage site and ending with a HpaI restriction endonuclease cleavage site.
As stated previously, the 1975 Lancefield study showed that the Ibc antigen was an effective vaccine antigen in a mouse model of Group B streptococcal infection (Lancefield, R. C., et al., J. Exp. Med. 142:165-179 (1975)). It was not clear at the time whether the alpha or beta protein component of the Ibc antigen was responsible for this protection. Madoff et al., began to shed light on this question and demonstrated that the purified C.beta. protein used as a vaccine could protect infant mice from experimental infection with Group B streptococci expressing this protein (Madoff, L. C., et al., Infect. Immun. 60:4989-4994 (1992)). Madoff et al., then went on to show that when they coupled a Type III streptococcal capsular polysaccharide to the C.beta. protein, producing a conjugate vaccine, this vaccine would protect infant mice against infection with either a Type III Group B streptococci (expressing no C.beta.) or a Type Ib Group B streptococci (expressing C.beta. but lacking a Type III capsular polysaccharide) (Madoff, L. C., et al., J. Clin. Invest. 94:286-292 (1994)). Thus, such a C.beta. protein conjugate vaccine served several functions: the polysaccharide elicited protective antibodies to the polysaccharide capsule and the C.beta. protein evoked protective antibodies to the protein as well as modified the immune response to the polysaccharide from a T independent response to a T dependent response.
This polysaccharide-C.beta. protein conjugate strategy works well in mice. But clearly, the goal is to protect humans against Group B streptococcal infections. The only caveat with using the same strategy in humans is that the C.beta. protein binds human IgA immunoglobulins non-specifically (C.beta. does not bind mouse IgA). This human IgA binding activity of C.beta. could diminish the efficacy of a polysaccharide-C.beta. protein conjugate vaccine for humans, as antigens bound to IgA can be cleared from the system so rapidly that an antigen-specific antibody response is not produced. Furthermore, potentially protective epitopes on the C.beta. protein could be hidden when the human IgA binds to the C.beta. molecule. Thus, it would be advantageous to obtain a mutant C.beta. protein which lacks the IgA binding capacity but retains as much of the native structure as possible.
With this goal in mind, several groups have attempted to determine the IgA binding region of the C.beta. protein. Jerlstrom et al. (Molec. Microbiol. 5:843-849 (1991)) used experiments wherein subfragments of the C.beta. protein were expressed as fusion proteins to identify two regions of the C.beta. protein capable of binding IgA. These experiments localized the IgA binding domains to a 747 bp BglII-HpaI fragment and a 1461 bp HpaI-HindIII fragment of the C.beta. protein. Furthermore, International Patent Application No. PCT/US/06111 describes the isolation of a C.beta. protein bearing a deletion of a region that binds IgA.